MLL is a histone methyltransferase whose mutation plays a critical role in leukemia pathogenesis and has a wide impact on patient morbidity and mortality. The studies in this application address the hypothesis that MLL is a bi-functional protein, which maintains or represses transcription depending on cellular conditions, subunit composition, and other unknown regulatory factors. Leukemogenic mutations are proposed to prevent this transcriptional inter-conversion required for extinction of target gene expression and subsequent terminal blood cell differentiation. This hypothetical model will be investigated using genetic, biochemical and molecular experimental approaches in four specific aims. In the first, two novel lines of mice with substitution mutations at the processing sites of MLL that result in non-cleavable or constitutively cleaved MLL proteins, respectively, will be employed to study the effect of proteolytic processing on MLL function during embryonic development and hematopoietic differentiation. Studies in the second aim will interrogate the compositions of MLL higher-order protein complexes using a combination of conventional and affinity purification techniques to establish the role of proteolytic processing and recently identified novel associated factors, such as HCF-1 and hASH2, in modulating the transcriptional effector properties of MLL. Together, studies in the first two aims will establish the critical stages in hematopoietic cell differentiation that depend on the hypothesized MLL biochemical transition and identify molecular mechanisms that orchestrate and regulate this process. Studies in the third and fourth aims will investigate critical accessory factors and subordinate pathways employed by MLL oncoproteins to initiate and sustain leukemogenesis. Aberrant recruitment of chromatin remodeling factors through MLL fusion partners will be assessed as a general mechanism for MLL oncogenic activation using genetic and biochemical techniques in combination with biologically relevant functional assays for tissue-specific oncogenesis. Similar experimental approaches will address whether there is a broad or highly selective requirement for Hox genes and Hox cofactors in MLL-mediated transformation. These overall efforts will identify mechanisms and molecules for understanding leukemia pathogenesis and provide candidate targets for rational drug design.